Project Summary Broadly neutralizing antibodies (bnAbs) recognize conserved epitopes on the envelope glycoprotein (Env) of the human immunodeficiency virus type-1 (HIV-1). Atomic structures of the BG505 SOSIP.664 gp140 trimer have provided a rational framework for Env-based HIV-1 vaccine design. The Wyatt group recently developed a cleavage-independent, native flexibly linked (NFL) trimer, which offers a promising alternative for Env-based vaccine design. Vaccine delivery is a critical issue yet to be addressed in developing well-folded trimers toward vaccine products. In a recent review, Schiller et al. promoted a virus-like particle (VLP) approach for HIV-1 vaccine delivery. Other delivery systems include mRNA and virus vectors. For the NFL trimer, it remains unclear which delivery system will produce the most robust immune response to enable protection against HIV- 1 infection. We hypothesize that a quantitative readout of B-cell response will facilitate rational evaluation of vaccine candidates since a robust cross-neutralizing antibody response is expected for an effective HIV-1 vaccine. Next-generation sequencing (NGS) has been widely used to study the diversity and maturation of bnAbs. We have developed a series of novel NGS technologies for the analysis of dynamic B-cell responses in natural infection, animal immunization, and human vaccine trials. In Project 3 of this HIVRAD application, we will investigate the Env-specific B-cell response to four vaccines based on the NFL trimer and different delivery systems in various animal models ? inbred mice, VelocImmune mice, rabbits and non-human primates (NHPs). In Aim 1, we will deep sequence the B-cell response of inbred mice, VelocImmune mice, and rabbits immunized with four vaccines. We will characterize the basic repertoire properties such as germline gene usage, degree of SHM, and CDR3 length and identify distinctive repertoire patterns associated with each vaccine. For each animal model, we will compare the B-cell responses to four vaccines. We will compare the B-cell responses in hu-mice and human vaccine trials. In Aim 2, we will deep sequence the B-cell response in NHPs immunized with four vaccines. Similarly, we will characterize the basic repertoire properties and identify distinctive patterns associated with four vaccines in the NHP model. We will compare the B-cell responses to four vaccines and compare the B-cell responses in NHPs and human vaccine trials. In Aim 3, we will study the developmental pathways of Env-specific B-cell lineages elicited by four vaccines. We will trace the lineages of monoclonal antibodies (mAbs) identified by single B-cell sorting (Project 2, Wyatt) and microfluidics-based single-cell analysis (Project 3, Zhu) in NGS repertoires. We will investigate whether the vaccine-elicited neutralizing mAbs resemble known bnAbs and their developmental pathways. Project 3, together with Project 1 (RNA and VSV vector), Project 2 (NFL trimer, VLP, and serum Ab analysis), and two Cores on in-vivo study and gene expression profiling, will constitute a comprehensive HIVRAD program towards an effective HIV-1 vaccine.